1. The Molecular Basis of Inheritance

2. What Is the Evidence that the Gene Is DNA? By the 1920s, it was known that chromosomes consisted of DNA and proteins. A new dye stained DNA and provided circumstantial evidence that DNA was the genetic material: It was in the right place It varied among species It was present in the right amount

3. Evidence That DNA Can Transform Bacteria Frederick Griffith in 1928 discovered genetic material he worked with two strains of a bacterium (Streptococcus pneumoniae), one pathogenic and one harmless.

4. When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic. He called this phenomenon transformation - now defined as a change in genotype and phenotype due to assimilation of foreign DNA.

5. Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS EXPERIMENT

6. In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA. Based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria. Oswald Avery Treated samples to destroy different molecules; if DNA was destroyed, the transforming principle was lost.

9. … more evidence that DNA is the genetic material Evidence that Viral DNA can program cells Hershey-Chase Experiment T2 phage and E.coli Either protein coat OR DNA will infect cell Tagged coat and DNA with radioactive markers Showed that the nucleic acid inside virus is transmitted to host cell

10. Hershey-Chase animation

13. What is the Structure of DNA? The structure of DNA was determined using many lines of evidence. One crucial piece came from X-ray crystallography. A purified substance can be made to form crystals; position of atoms is inferred by the pattern of diffraction of X-rays passed through it.

14. Rosalind Franklin With X-ray Crystallography they knew Shape Width Spacing between nucleotides

15. Chemical composition also provided clues DNA is a polymer of nucleotides: deoxyribose, a phosphate group, and a nitrogen-containing base. The bases: Purines: adenine (A), guanine (G) Pyrimidines: cytosine (C), thymine (T)

16. Nucleotides Have Three Components repeat fig 3.23 here

17. 1950 - Erwin Chargaff found in the DNA from many different species: amount of A = amount of T amount of C = amount of G Or, the abundance of purines = the abundance of pyrimidines — Chargaff’s rule.

18. Chargaff’s Rule

19. Model building started by Linus Pauling — building 3-D models of possible molecular structures. Francis Crick and James Watson used model building and combined all the knowledge of DNA to determine its structure. Watson animation

21. X-ray crystallography convinced them the molecule was helical. Other evidence suggested there were two polynucleotide chains that ran in opposite directions — antiparallel. 1953 — Watson and Crick established the general structure of DNA.

22. Key features of DNA A double-stranded helix, uniform diameter It is right-handed It is antiparallel Outer edges of nitrogenous bases are exposed in the major and minor grooves

23. Complementary base pairing Adenine pairs with thymine by two hydrogen bonds. Cytosine pairs with guanine by three hydrogen bonds. Every base pair consists of one purine and one pyrimidine.

24. Purines and Pyrimidines Pyrimidine Purine Nucleotide

27. Antiparallel strands: direction of strand is determined by the sugar – phosphate bonds. Phosphate groups connect to the 3′ C of one sugar, and the 5′ C of the next sugar. At one end of the chain — a free 5′ phosphate group; at the other end a free 3′ hydroxyl.

28. Functions of DNA Store genetic material — millions of nucleotides; base sequence stores and encodes huge amounts of information Susceptible to mutation — change in information

29. Genetic material is precisely replicated in cell division — by complementary base pairing. Genetic material is expressed as the phenotype — nucleotide sequence determines sequence of amino acids in proteins.

30. How is DNA Replicated? The DNA is a template for the synthesis of new DNA Three possible replication patterns: Semiconservative replication Conservative replication Dispersive replication

31. semiconservative model - predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)

32. Three Models for DNA Replication Semiconservative Conservative Dispersive semiconservative DNA replication animation

33. Meselson and Stahl showed that semiconservative replication was the correct model. They used density labeling to distinguish parent DNA strands from new DNA strands. DNA was labeled with 15 N, making it more dense.

36. Results of their experiment can only be explained by the semiconservative model. If it was conservative, the first generation of individuals would have all been high or low density, but not intermediate. If dispersive, density in the first generation would be half, but this density would not appear in subsequent generations.

37. Two Steps in DNA Replication The double helix is unwound, making two template strands. New nucleotides are added to the new strand at the 3′ end; joined by phosphodiester linkages. Sequence is determined by complementary base pairing.

38. Each New DNA Strand Grows from Its 5′ End to Its 3′ End

40. A large protein complex — the replication complex — catalyzes the reactions of replication. All chromosomes have a base sequence called origin of replication (ori). Replication complex binds to ori at the start. DNA replicates in both directions, forming two replication forks.

41. Two Views of DNA Replication

42. DNA helicase uses energy from ATP hydrolysis to unwind the DNA. Single-strand binding proteins keep the strands from getting back together.

43. Small, circular chromosomes have a single origin of replication. As DNA moves through the replication complex, two interlocking circular chromosomes are formed. DNA topoisomerase separates the two chromosomes.

44. Replication in Small Circular Chromosomes

45. Large linear chromosomes have many origins of replication. DNA is replicated simultaneously at the origins.

46. DNA polymerases are much larger than their substrates. Shape is like a hand; the “finger” regions have precise shapes that recognize the shapes of the nucleotide bases.

47. A primer is required to start DNA replication — a short single strand of RNA. Primer is synthesized by primase. Then DNA polymerase begins adding nucleotides to the 3′ end of the primer.

48. No DNA Forms without a Primer

49. Cells have several DNA polymerases. One is for DNA replication; others are involved in primer removal and DNA repair. Other proteins are involved in the replication process.

50. At the replication fork The leading strand is pointing in the “right” direction for replication. The lagging strand is in the “wrong” direction. Synthesis of the lagging strand occurs in small, discontinuous stretches — Okazaki fragments.

51. The Two New Strands Form in Different Ways Leading / lagging strand synthesis animation

52. Each Okazaki fragment requires a primer. The final phosphodiester linkage between fragments is catalyzed by DNA ligase.

55. DNA polymerases work very fast: They are processive - catalyze many polymerizations each time they bind to DNA Newly replicated strand is stabilized by a sliding DNA clamp (a protein)

56. A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization

57. The new chromosome has a bit of single stranded DNA at each end (on the lagging strand) — this region is cut off. Eukaryote chromosomes have repetitive sequences at the ends called telomeres.

58. Telomeres and Telomerase

59. Human chromosome telomeres (TTAGGG) are repeated about 2500 times. Chromosomes can lose 50 – 200 base pairs with each replication. After 20 – 30 divisions, the cell dies.

60. Some cells — bone marrow stem cells, gamete-producing cells — have telomerase that catalyzes the addition of telomeres. 90% of human cancer cells have telomerase; normal cells do not. Some anticancer drugs target telomerase.

61. How are Errors in DNA Repaired? DNA polymerases make mistakes in replication, and DNA can be damaged in living cells. Repair mechanisms: Proofreading Mismatch repair Excision repair

62. As DNA polymerase adds a nucleotide to a growing strand, it has a proofreading function — if bases are paired incorrectly, the nucleotide is removed.

63. The newly replicated DNA is scanned for mistakes by other proteins. Mismatch repair mechanism detects mismatched bases — the new strand has not yet been modified (e.g., methylated in prokaryotes) so it can be recognized. If mismatch repair fails, the DNA is altered.

64. DNA can be damaged by radiation, toxic chemicals, and random spontaneous chemical reactions. Excision repair: enzymes constantly scan DNA for mispaired bases, chemically modified bases, and extra bases — unpaired loops.